O-vanillin derived schiff bases and their organotin(Iv) compounds: Synthesis, structural characterisation, in-silico studies and cytotoxicity

Article abstract of DOI:10.3390/ijms20040854

Six new organotin(IV) compounds of Schiff bases derived from S-R-dithiocarbazate [R = benzyl (B), 2-or 4-methylbenzyl (2M and 4M, respectively)] condensed with 2-hydroxy-3-methoxybenzaldehyde (oVa) were synthesised and characterised by elemental analysis, various spectroscopic techniques including infrared, UV-vis, multinuclear (¹H,¹³C,¹¹⁹Sn) NMR and mass spectrometry, and single crystal X-ray diffraction. The organotin(IV) compounds were synthesised from the reaction of Ph₂SnCl₂ or Me₂SnCl₂ with the Schiff bases (S2MoVaH/S4MoVaH/SBoVaH) to form a total of six new organotin(IV) compounds that had a general formula of [R₂Sn(L)] (where L = Schiff base; R = Ph or Me). The molecular geometries of Me₂Sn(S2MoVa), Me₂Sn(S4MoVa) and Me₂Sn(SBoVa) were established by X-ray crystallography and verified using density functional theory calculations. Interestingly, each experimental structure contained two independent but chemically similar molecules in the crystallographic asymmetric unit. The coordination geometry for each molecule was defined by thiolate-sulphur, phenoxide-oxygen and imine-nitrogen atoms derived from a dinegative, tridentate dithiocarbazate ligand with the remaining positions occupied by the methyl-carbon atoms of the organo groups. In each case, the resulting five-coordinate C₂NOS geometry was almost exactly intermediate between ideal trigonal-bipyramidal and square-pyramidal geometries. The cytotoxic activities of the Schiff bases and organotin(IV) compounds were investigated against EJ-28 and RT-112 (bladder), HT29 (colon), U87 and SJ-G2 (glioblastoma), MCF-7 (breast) A2780 (ovarian), H460 (lung), A431 (skin), DU145 (prostate), BE2-C (neuroblastoma) and MIA (pancreatic) cancer cell lines and one normal breast cell line (MCF-10A). Diphenyltin(IV) compounds exhibited greater potency than either the Schiff bases or the respective dimethyltin(IV) compounds. Mechanistic studies on the action of these compounds against bladder cancer cells revealed that they induced the production of reactive oxygen species (ROS). The bladder cancer cells were apoptotic after 24 h post-treatment with the diphenyltin(IV) compounds. The interactions of the organotin(IV) compounds with calf thymus DNA (CT-DNA) were experimentally explored using UV-vis absorption spectroscopy. This study revealed that the organotin(IV) compounds have strong DNA binding affinity, verified via molecular docking simulations, which suggests that these organotin(IV) compounds interact with DNA via groove-binding interactions.

Full text of DOI:10.3390/ijms20040854

International Journal of
Molecular Sciences
Article
o-Vanillin Derived Schiff Bases and Their
Organotin(IV) Compounds: Synthesis, Structural
Characterisation, In-Silico Studies and Cytotoxicity
Enis Nadia Md Yusof 1
,2
, Muhammad A. M. Latif 1 , Mohamed I. M. Tahir ,
1
Jennette A. Sakoff , Michela I. Simone , Alister J. Page * , Abhi Veerakumarasivam 5,6,
Edward R. T. Tiekink 7 and Thahira B. S. A. Ravoof 1,8,
3
2,4
2,
*
1
Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang,
Selangor Darul Ehsan, Malaysia; enisnadia89@gmail.com (E.N.M.Y.); aliflatif@upm.edu.my (M.A.M.L.);
ibra@upm.edu.my (M.I.M.T.)
2
3
4
5
6
7
8
Discipline of Chemistry, School of Environmental and Life Sciences, University of Newcastle,
University Drive, Callaghan, NSW 2308, Australia; michela.simone@newcastle.edu.au
Experimental Therapeutics Group, Department of Medical Oncology, Calvary Mater Newcastle Hospital,
Edith Street, Waratah NSW 2298, Australia; jennette.sakoff@newcastle.edu.au
Priority Research Centre for Chemical Biology & Clinical Pharmacology, University of Newcastle,
University Drive, Callaghan, NSW 2308, Australia
Department of Biological Sciences, School of Science and Technology, Sunway University,
No. 5 Jalan Universiti, 47500 Bandar Sunway, Selangor Darul Ehsan, Malaysia; abhiv@sunway.edu.my
Medical Genetics Laboratory, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia,
4
3400 UPM Serdang, Selangor Darul Ehsan, Malaysia
Research Centre for Crystalline Materials, School of Science and Technology, Sunway University,
No. 5 Jalan Universiti, 47500 Bandar Sunway, Selangor Darul Ehsan, Malaysia; edward.tiekink@gmail.com
Materials Synthesis and Characterization Laboratory, Institute of Advanced Technology,
Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia
*
Correspondence: Correspondence: alister.page@newcastle.edu.au (A.J.P.); thahira@upm.edu.my (T.B.S.A.R.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 5 December 2018; Accepted: 30 December 2018; Published: 15 February 2019
Abstract: Six new organotin(IV) compounds of Schiff bases derived from S-R-dithiocarbazate
R = benzyl (B), 2- or 4-methylbenzyl (2M and 4M, respectively)] condensed with 2-hydroxy-3-
methoxybenzaldehyde (oVa) were synthesised and characterised by elemental analysis, various
[
1
13
119
spectroscopic techniques including infrared, UV-vis, multinuclear ( H, C, Sn) NMR and mass
spectrometry, and single crystal X-ray diffraction. The organotin(IV) compounds were synthesised
from the reaction of Ph SnCl or Me SnCl with the Schiff bases (S2MoVaH/S4MoVaH/SBoVaH)
2
2
2
2
to form a total of six new organotin(IV) compounds that had a general formula of [R Sn(L)] (where
2
L = Schiff base; R = Ph or Me). The molecular geometries of Me Sn(S2MoVa), Me Sn(S4MoVa) and
2
2
Me Sn(SBoVa) were established by X-ray crystallography and verified using density functional theory
2
calculations. Interestingly, each experimental structure contained two independent but chemically
similar molecules in the crystallographic asymmetric unit. The coordination geometry for each
molecule was defined by thiolate-sulphur, phenoxide-oxygen and imine-nitrogen atoms derived
from a dinegative, tridentate dithiocarbazate ligand with the remaining positions occupied by the
methyl-carbon atoms of the organo groups. In each case, the resulting five-coordinate C NOS
2
geometry was almost exactly intermediate between ideal trigonal-bipyramidal and square-pyramidal
geometries. The cytotoxic activities of the Schiff bases and organotin(IV) compounds were
investigated against EJ-28 and RT-112 (bladder), HT29 (colon), U87 and SJ-G2 (glioblastoma), MCF-7
(breast) A2780 (ovarian), H460 (lung), A431 (skin), DU145 (prostate), BE2-C (neuroblastoma) and
MIA (pancreatic) cancer cell lines and one normal breast cell line (MCF-10A). Diphenyltin(IV)
compounds exhibited greater potency than either the Schiff bases or the respective dimethyltin(IV)
compounds. Mechanistic studies on the action of these compounds against bladder cancer cells
Int. J. Mol. Sci. 2019, 20, 854; doi:10.3390/ijms20040854
www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2019, 20, 854
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revealed that they induced the production of reactive oxygen species (ROS). The bladder cancer
cells were apoptotic after 24 h post-treatment with the diphenyltin(IV) compounds. The interactions
of the organotin(IV) compounds with calf thymus DNA (CT-DNA) were experimentally explored
using UV-vis absorption spectroscopy. This study revealed that the organotin(IV) compounds have
strong DNA binding affinity, verified via molecular docking simulations, which suggests that these
organotin(IV) compounds interact with DNA via groove-binding interactions.
Keywords: tridentate ONS Schiff bases; organotin(IV); dithiocarbazate; five-coordinate compounds;
single-crystal X-ray diffraction analysis; cytotoxic activity; mechanistic studies; molecular docking
1
. Introduction
The International Agency for Research on Cancer reported a global incidence of 18.1 million new
cancer cases and 9.6 million deaths in 2018, with the number of new cases expected to increase rapidly
over the next two decades [ ]. Chemotherapy remains the most widely-used cancer treatment,
1,2
as compared to surgery, radiation therapy, immunotherapy, hormone therapy and targeted therapy.
Cisplatin is a long-standing effective chemotherapy agent for a number of cancers including testicular,
ovarian, cervical, head and neck, bladder, lung and colorectal cancer [
such as carboplatin, oxaliplatin, nedaplatin and laboplatin are also effective chemotherapeutic agents
against tumours [ ]. A probable mechanism of action for cisplatin is via the interaction with DNA
though the formation of covalent adducts [ ]. However, platinum drugs have been known to
cause various side-effects including anaemia, diarrhoea, alopecia, petechiae, fatigue, nephrotoxicity,
emetogenesis, ototoxicity, neurotoxicity and increasing chemotherapeutic resistance [ ]. This has
3,4]. Platinum-based compounds
5
4
6
sparked wide-spread research in the synthesis and evaluation of novel non-platinum-based complexes
as alternative chemotherapeutic agents.
In recent years, metal compounds with a stable d10 electronic configuration have received
considerable attention due to the wide variety of structures formed that depend on the coordinating
ligands and synthetic procedures [7–16]. Organotin(IV) compounds have found use in industry,
medicine and agriculture as stabilisers in polyvinylchloride (PVC) plastics, catalysts [17], anti-fouling
paints, fungicides [10], wood preservatives and disinfectants [18]. Organotin(IV) compounds have
also been reported as effective biocidal agents against environmental toxicants [19
medicinal applications (such as anti-tumour, anti-microbial, anti-nematicidal, anti-insecticidal and
anti-inflammatory agents) have been explored [ 21 23]. The National Cancer Institute (NCI) has
,20]. Their
7, –
tested over 2000 tin-based pharmacological candidates, the largest number ever tested among the
metal-containing drug candidates [24].
Many mechanistic studies on organotin(IV) compounds have indicated that these compounds
interact with DNA [25
activity of organotin(IV) compounds is believed to arise from their tendency in binding to or cleaving
DNA [27 28]. Wang et al. reported that organotin(IV) compounds containing Schiff bases could
interact with CT-DNA through intercalation interactions of cytotoxic drugs and serum albumin
which is involved in the transportation of compounds through the blood stream), where the nature
and magnitude of these interactions that could significantly influence the pharmacokinetics have
also been investigated [29 30]. Organotin(IV) derivatives bind to both nitrogenous DNA bases and
phosphate-oxygen of the DNA sugar backbone [31 32]. Organotin(IV) compounds have been reported
,26], one of the main targets in designing cytotoxic drugs and the biological
,
(
,
,
to bind to glycoprotein or cellular proteins, hence interacting directly with DNA, causing cell death.
Tin(IV) compounds have also been found to induce DNA damage by specifically binding to the
phosphate backbone of DNA thus leading changes in DNA conformations [33].
The activity of organotin(IV) compounds can be readily modified by the choice of ligand
coordinated to the central Sn(IV) ion. Organotin(IV) centres coordinated to hetero-donor atoms,

Int. J. Mol. Sci. 2019, 20, 854
3 of 34
especially oxygen, nitrogen and sulphur, have been investigated with a particular focus on their
structure-activity correlations [34 36]. Coordination of metals to multidentate Schiff bases have, in
most cases, enhanced the biological activity of these compounds [37 39]. Tin(IV) compounds of Schiff
–
–
bases containing pyridine have been reported to be particularly biologically relevant [38,40,41].
In order to expand therapeutic potential of organotin(IV) compounds of dithiocarbazate
derivatives, and in light of the biological relevance of previously reported organotin(IV) compounds,
we report herein the synthesis of three Schiff bases derived from 2-hydroxy-3-methoxybenzaldehyde
dithiocarbazate and six of their organotin(IV) compounds. The X-ray crystal structures of
dimethyltin(IV) compounds were determined, and their geometries were compared with theoretical
DFT calculations at the Becke, 3-parameter, Lee–Yang–Parr (B3LYP) level of theory. The Schiff bases and
organotin(IV) compounds were tested for their cytotoxicity via various assays, which was supported
by DNA binding studies.
2
. Results and Discussion
2
.1. Synthesis
Schiff bases, S2MoVaH, S4MoVaH and SBoVaH, were prepared by reacting S-N-R-
benzyldithiocarbazates (N = 2,3, R = methyl) and oVa in equimolar ratios (Scheme 1). The Schiff
bases were obtained in good yields. The organotin(IV) compounds were synthesised using the
corresponding diorganotin(IV) dichloride salts under reflux as shown in Scheme 2. The Schiff bases
and their organotin(IV) compounds are yellow, stable at room temperature and soluble in organic
solvents (especially chloroform, dimethyl sulfoxide (DMSO) and dimethylformamide (DMF). The room
temperature molar conductance values of the organotin(IV) compounds in DMSO at a concentration
−
3
−1
2
−1
−1
2
−1
of 10 M were in the range of 2.30–8.21
Ω
·
cm
·
mol , i.e., values well below 25
Ω
·
cm
·
mol ,
indicating that these organotin(IV) compounds are non-electrolytes proving the absence of counter-ions,
thus indicating that the dithiocarbazate Schiff bases were covalently bonded to the central organotin
ion [42].
Scheme 1. Synthetic pathway for the formation of SBoVaH, S2MoVaH, and S4MoVaH.

Int. J. Mol. Sci. 2019, 20, 854
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Scheme 2. Synthesis of organotin(IV) compounds.
2
.2. IR Spectral Analysis
The IR spectra indicated the successful formation of the dithiocarbazate Schiff bases and their
organotin(IV) compounds. Infrared spectra of Schiff bases and their organotin(IV) compounds were
−
1
measured in the range 4000 to 280 cm and verified using frequencies predicted using density
functional theory (DFT). All DFT vibrational frequencies were scaled using a scaling factor of 0.9682 [43
see Section 3.5.1 for full details of DFT calculations). The Schiff bases have a thioamide(–NH–CS–)
]
(
functional group and thus, can exist either in the thione (Figure 1a) or thiol (Figure 1b) forms or as a
mixture of both thione and thiol tautomeric forms. In the solid-state, the Schiff bases are primarily
−
1
in the thione form as shown by the presence of the v(NH) band at 3089 cm and absence of the
v(SH) band at c.a. 2600 cm
−
1
[44]. Characteristic vibrational frequencies consistent with the successful
formation of organotin(IV) compounds have been identified. The v(NH) stretching vibration at ca
−
1
3
084–3092 cm was absent in the organotin(IV) compounds indicating that the Schiff bases were
deprotonated upon complexation. Sharp v(C=N) bands corresponding to the Schiff base azomethine
−
1
group at 1600 (S2MoVaH), 1598 (S4MoVaH) and 1598 (SBoVaH) cm were observed to shift to lower
wavenumbers upon complexation which indicated coordination via the azomethine nitrogen [45].
−
1
The v(CSS) splitting bands found in the range 956–964 cm provide strong evidence of coordination
of the thiolate sulphur atom to the tin ion. The shifting of the hydrazinic v(N–N) band to lower
wavenumbers in the organotin(IV) compounds showed evidence of coordination via the azomethine
nitrogen due to reduction in the repulsion between lone pairs of electrons on the nitrogen atom [46–48].
The calculated frequencies correlated well with the experimental frequencies observed in the FT-IR
spectra, considering that DFT frequency calculations were performed in the gas-phase. The N–H
band in the experimental FTIR spectra of Schiff bases red-shifted owing to intermolecular hydrogen
bonding [49]. The absence of hydroxyl stretching in the experimental spectra of the Schiff bases could
be due to intermolecular and intramolecular hydrogen bonding as was observed in similar reported

Int. J. Mol. Sci. 2019, 20, 854
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organotin(IV) compounds [50]. Moreover, only slight differences were observed in the experimental
and calculated data for v(C=N), v(N–N) and v(C=S). Complete IR data are provided in Table S1.
Figure 1. The thione (a) and thiol (b) tautomeric forms of the Schiff bases (R = o–CH , S2MoVaH;
3
p-CH , S4MoVaH; R = H, SBoVaH).
3
2
.3. NMR Spectroscopic Analysis
NMR spectra of the dithiocarbazate Schiff bases were recorded in DMSO-d , while the NMR
6
spectra of their organotin(IV) compounds were recorded in CDCl . The data obtained were in good
3
agreement with the expected structures in both solvents. The absence of the exchangeable SH signal
1
in the H NMR spectra of the Schiff bases indicated the predominance of the thione tautomer in
DMSO-d6 [51]. The spectra of the Schiff bases also exhibited a downfield signal at ~13.32–13.34 ppm
1
due to the NH group. For all organotin(IV) compounds, the H NMR signal for the azomethine NH
proton was absent due to the coordination to the metal ion by the Schiff base, in corroboration with the
absence of the v(NH) FTIR band noted in Section 2.2. A singlet appeared in the downfield region of
1
2
the H NMR spectra of the Schiff bases which corresponded to an sp -C–O
H proton. Proton chemical
shifts for both NH and OH disappeared in the spectra of the organotin(IV) compounds suggesting
double deprotonation of the Schiff bases and supporting coordination of both the phenolic-oxygen
and -nitrogen atoms of the Schiff base to the tin ion.
The 13C NMR spectra of the dithiocarbazate Schiff bases showed a downfield chemical shift at
~196 ppm, indicating the existence of the C=S group and thus the predominance of the thione tautomer
in solution. This signal was shifted upfield in the organotin(IV) compounds (170–180 ppm), indicating
a decrease in electron density at the C=S carbon atom when the sulphur atom was complexed to the
tin ion, consistent with the structures proposed for the organotin(IV) compounds [52].
The 1 Sn NMR spectra of the organotin(IV) compounds were recorded at room temperature in
19
CDCl using SnMe as the external standard. Theoretically, as the coordination number about tin
3
4
119
119
increases, chemical shifts in Sn NMR move towards lower frequencies [53].
Sn NMR spectra are
also very sensitive to the type of donor atom coordinated to the tin and its environment, e.g., alkyl
or aryl groups in the case of a C donor and to the bond angles around the tin centre [54]. However,
shielding or deshielding of the tin nucleus does not have a significant effect on the chemical shifts [55].
Dimethyltin(IV) compounds showed less negative 1 Sn chemical shifts compared to diphenyltin(IV)
compounds. The chemical shift values obtained were within the range reported for other diphenyl- or
19
dimethyl(IV) compounds with heterocyclic ligands having penta-coordinated trigonal bipyramidal
56]. Complete 1 Sn NMR data are provided in Section 3.3. The NMR details for the
19
geometry [53
,
Schiff bases and organotin(IV) compounds are tabulated in Tables S2 and S3.
2
.4. Mass Spectral Analysis
Mass spectral data for the Schiff bases were recorded in DMSO and were found to be consistent
with the proposed formulation of the Schiff bases. Mass spectra displayed prominent peaks at
+
m/z 346.15 and 346.10 for S2MoVaH and S4MoVaH, respectively, corresponding to [C H N O S ] ,
17
18
2
2 2
+
and m/z 332.10 for SBoVaH corresponding to [C H N O S ] .
16
16
2
2 2

Int. J. Mol. Sci. 2019, 20, 854
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2
.5. X-ray Crystallography
The crystal structures of Me Sn(S2MoVa), Me Sn(S4MoVa) and Me Sn(SBoVa), each featured two
2
2
2
independent molecules in the crystallographic asymmetric unit, i.e., molecules a and b. Figure 2a–c
shows the molecular structures for the first independent molecule in each crystal and images for the
second independent molecules are shown in Figures 3a, 4a and 5a; selected geometric parameters
are collated in Table 1. The major conformational difference between the molecules in each of
Me Sn(S2MoVa), Me Sn(S4MoVa) and Me Sn(SBoVa) is highlighted in the overlay diagram shown in
2
2
2
Figure 2d. Whereas in the tolyl species, the tolyl substituent is orientated to be directed away from the
coordinated S1 atom, i.e., can be considered anti to S1, in each molecule of Me Sn(S4MoVa), the phenyl
2
ring is syn to the S1 atom.
The tin atom in Me Sn(S2MoVa) is coordinated by the thiolate-S1, phenoxide-O1 and imine-N
2
atoms derived from the dinegative, [(3-methoxy-2-oxyphenyl)methyl] dithiocarbazate ligand.
The five-coordinate geometry is completed by a carbon atom from each of the two methyl groups.
The assignment of thiolate-S1 is readily made by comparing the length of the C1-S1 bond in
Me Sn(S2MoVa), i.e., 1.726(9) and 1.717(8) Å, for the two independent molecules comprising the
2
asymmetric unit, with that in the uncoordinated Schiff base (which lacks the 2-methyl group in
the ester group) of 1.670(2) Å [50], i.e., the bond lengths in Me Sn(S2MoVa) are significantly longer.
2
The C1-S2 bond lengths are experimentally equivalent at 1.744(8) and 1.747(8) Å [50]. However, the two
C1=N1 imine bond lengths in Me Sn(S2MoVa) are 1.304(11) and 1.273(10) Å, which are significantly
2
longer than the equivalent C1–N1(H) bond of 1.338(2) Å in the free Schiff base [50]. The mode of
coordination of the dithiocarbazate ligand leads to the formation of five- and six-membered chelate
◦
rings, with the chelate angle in the former being approximately 5 more acute than that in the latter,
Table 1. The Sn, S1, N1, N2, C1 chelate ring in molecule a is non-planar and is twisted about the Sn-S1
bond. On the other hand, the Sn, O1, N1, C10–C12 chelate ring is best described as having an envelope
conformation whereby the Sn atom lies 0.528(11) Å above the plane. However, the structure of the
second independent molecule in the asymmetric unit, molecule b, is slightly different whereby the
five-membered ring is an envelope with the Sn atom lying 0.372(12) Å above the plane defined by
the remaining four atoms in this ring [r.m.s. deviation = 0.008 Å]. The six-membered ring is also an
envelope with the Sn atom lying 0.528(11) Å out of the plane defined by the five remaining atoms which
present a r.m.s. deviation = 0.022 Å. The dihedral angle between the best planes through the chelate
◦
◦
rings in the molecule a is 11.12(17) while for molecule b, the equivalent angle is 12.4(3) . The benzyl
ring is almost orthogonal to the five-membered chelate ring for both independent molecules in the
◦
unit cell, forming dihedral angles of 88.3(3) and 86.8(3) , respectively. Similarly, the dihedral angles
between the outer rings are 72.3(3) and 75.5(3) , respectively, also indicating an approximate orthogonal
◦
relationship. The similarity between the conformations found for the two independent molecules in
the asymmetric unit is highlighted in the overlay diagram shown in Figure 3. The five-coordinate
geometry defined by the C NOS donor set for molecule a, with five-coordinate geometry descriptor
2
τ = 0.48, is intermediate between ideal square-pyramidal (
τ = 0.0) and trigonal-bipyramidal geometries
(τ
= 1.0) [57]; the value of is 0.50 for molecule b. For each independent molecule, the widest angle
τ
corresponds to the S1-Sn-O1 angle with the second widest angle being subtended by the tin-bound
◦
methyl groups. The angles about the quaternary-C1 atom span nearly 20 with the widest angles
involving the doubly bonded N1 atom. Of these, the angle involving the S1 atom, being the widest,
is consistent with some double bond character in the C1-S1 bond. Consistent with this, (see Table 1),
the C1-S1 bond lengths are systematically shorter than the C1-S2 bonds.

Int. J. Mol. Sci. 2019, 20, 854
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Figure 2. Molecular structures of the first independent molecules of (a) Me Sn(S2MoVa),
2
(b
) Me Sn(S4MoVa) and (
c
) Me Sn(SBoVa) showing atom labelling schemes. (
d) Overlay diagram of the
2
2
two independent molecules of each of Me Sn(S2MoVa), a (red image) and b (green), Me Sn(S4MoVa),
2
2
a (blue) and b (pink), and Me Sn(SBoVa) a (yellow) and b (aqua) with the SnMe planes superimposed.
2
2

Int. J. Mol. Sci. 2019, 20, 854
8 of 34
Figure 3. Crystallographic diagrams for Me Sn(S2MoVa): (
a
) Molecular structure of the second
) overlay diagram of molecules a (red image) and inverted-b
green) drawn so the SnC2 atoms of the Me Sn moiety are overlapped and ( ) a view of the
2
independent molecule, molecule b, (
b
(
c
2
supramolecular layer in the ab-plane (left image) and a view of the unit cell contents in projection down
the a-axis with one layer highlighted in space-filling mode (right image). The C–H . O and C–H
. .
. . .
π
interactions are shown as orange and purple dashed lines, respectively.
The isomeric compound, Me Sn(S4MoVa), having an ester 4-tolyl residue rather than
2
2
-tolyl, presents very similar characteristics to those exhibited in the structure of Me Sn(S2MoVa)
2
(Figures 2b and 4, Table 1). The conformations of the two chelate rings in the independent molecules
are the approximately same, being based on an envelope conformation with the tin atom being the flap
in all cases. The envelope is more pronounced, cf. Me Sn(S2MOVa), in the five-membered rings with
2
the tin atom lying 0.486(4) Å [0.490(4) Å for molecule b] above the plane of the four remaining atoms
which have a r.m.s. deviation = 0.009 Å [0.012 Å] but, is somewhat flattened in the six-membered
rings with the tin atom lying 0.187(4) Å [0.132(4) Å] out of the plane defined by the five remaining
atoms with a r.m.s. deviation = 0.013 Å [0.017 Å]. The dihedral angle between the best planes through
◦
◦
the chelate rings is 10.24(8) [8.53(11) ]. Near orthogonal relationships between the five-membered
chelate rings and the pendant tolyl rings persist, and the dihedral angles between the outer phenyl
◦
◦
rings is 65.75(10) [78.30(10) ]. The value of
τ is 0.54 [0.41]. A difference is noted in the coordination
geometries in that the second widest angles at the tin atom, after S1–Sn–O1, are subtended by the N2
◦
◦
and C19 atoms, i.e., 125.83(12) [131.09(12) ], rather than the C18–Sn–C19 angle, Table 1.

Int. J. Mol. Sci. 2019, 20, 854
9 of 34
Figure 4. Crystallographic diagrams for Me Sn(S4MoVa): (
a
) Molecular structure of the second
) overlay diagram of molecules a (blue image) and inverted-b
) a view of the
(chelate) interactions (left image; non-participating
2
independent molecule, molecule b, (
b
(
pink) drawn so the SnC2 atoms of the Me Sn moiety are overlapped and (c
2
supramolecular dimer sustained by C–H . . .
π
hydrogen atoms have been omitted) and a view of the unit cell contents in projection down the a-axis.
The C–H . . . O and C–H π interactions are shown as orange and purple dashed lines, respectively.
. . .
The unsubstituted analogue of the aforementioned isomeric structures, i.e., Me Sn(SBOVa), is
2
similar to those just described (Figures 2c and 5, Table 1). A likely trend in the Sn-S1 bond lengths may
be discerned, Table 1, in that these are longest in Me Sn(SBOVa) compared to the tolyl derivatives,
2
Me Sn(S2MOVa) and Me Sn(S4MOVa), pointing to an electronic influence, i.e., activation of the
2
2
aromatic ring owing to the presence of electron-donating methyl groups; while there is evidence
for shorter Sn-S1 bond lengths in Me Sn(S4MOVa) cf. Me Sn(S2MOVa), the standard uncertainty
2
2
values in the latter preclude a definitive statement on this although the trend for shorter Sn-S1
bonds in Me Sn(S4MOVa) is consistent with expectation. The conformational flexibility in the chelate
2
ligands formed by these tridentate ligands is reflected in the flattened envelope conformations for the
five-membered rings with the Sn atom lying 0.326(6) Å above the plane defined by the four remaining
atoms [r.m.s. deviation = 0.021 Å; comparable parameters for molecule b are 0.287(6) and 0.024 Å,
respectively]. By contrast, pronounced envelope conformations are found for the six-membered rings
[
0.675(5) and 0.043 Å for molecule a; 0.706(5) and 0.048 Å for molecule b]. The dihedral angle between
◦
◦
the best planes through the chelate rings is 13.66(16) [13.92(17) ]. The values of
τ for the independent
molecules compute to 0.47 and 0.44, respectively. As for Me Sn(S4MOVa), after the S1–Sn–O1 angles,
2
the widest angle subtended at the tin atom in Me Sn(SBOVa) for the second independent molecule
2
◦
involves the coordinated imine-N2 and a tin-bound methyl-C17 atoms, i.e., 127.66(17) . Of the three
new structures reported herein, the greatest agreement between the two independent molecules
comprising the asymmetric unit is found for Me Sn(S4MOVa), see Figure 5 and Table 1.
2

Int. J. Mol. Sci. 2019, 20, 854
10 of 34
◦
Table 1. Selected geometric parameters (Å, ) for Me Sn(S2MoVa), Me Sn(S4MoVa) and Me Sn(SBoVa).
2
2
2
Parameter
Me2Sn(S2MoVa)
Me2Sn(S4MoVa)
Me2Sn(SBoVa)
B3LYP
LanL2DZ/6-311G(d,p)
B3LYP
LanL2DZ/6-311G(d,p)
B3LYP
LanL2DZ/6-311G(d,p)
Bond lengths
molecule a
molecule b
molecule a
molecule b
molecule a
molecule b
Sn–S1
Sn–O1
Sn–N2
C1–S1
2.545(2)
2.092(6)
2.188(6)
1.726(9)
1.744(8)
1.394(9)
1.304(11)
1.308(9)
2.542(2)
2.102(6)
2.192(6)
1.717(8)
1.747(8)
1.406(9)
1.273(10)
1.306(9)
2.576
2.085
2.285
1.751
1.772
1.389
1.293
1.313
2.5391(8)
2.090(2)
2.223(2)
1.736(3)
1.752(3)
1.402(3)
1.289(4)
1.309(4)
2.5309(7)
2.214(2)
2.088(2)
1.734(3)
1.757(3)
1.409(3)
1.287(4)
1.307(4)
2.566
2.078
2.232
1.753
1.771
1.388
1.293
1.312
2.5789(11)
2.103(3)
2.194(3)
1.725(4)
1.771(4)
1.397(5)
1.298(5)
1.311(5)
2.5607(10)
2.093(3)
2.205(3)
1.726(4)
1.763(4)
1.395(4)
1.291(5)
1.304(5)
2.590
2.078
2.226
1.748
1.783
1.383
1.296
1.307
C1–S2
N1–N2
C1–N1
C(x)–N2
Bond angles
S1–Sn–O1
S1–Sn–N2
O1–Sn–N2
C18–Sn–C(y)
S1–C1–S2
157.89(17)
77.35(18)
82.2(2)
128.9(5)
112.1(5)
129.1(6)
118.9(7)
158.25(17)
76.94(18)
82.3(2)
128.2(5)
111.9(5)
128.9(6)
119.3(6)
159.5
77.6
82.0
123.9
113.4
128.2
118.4
158.51(7)
77.33(7)
82.13(9)
122.58(14)
111.46(17)
129.1(2)
155.88(7)
77.78(6)
82.15(8)
123.05(14)
111.81(17)
129.6(2)
159.7
77.8
82.2
123.9
113.4
128.2
118.4
155.82(9)
76.70(9)
82.76(11)
127.70(18)
120.2(2)
128.7(3)
111.1(3)
154.24(10)
77.13(8)
82.09(12)
124.8(2)
120.1(2)
128.3(3)
111.6(3)
159.8
77.3
82.4
123.7
120.8
127.7
111.5
S1–C1–N1
S2–C1–N1
119.5(2)
118.6(2)

Int. J. Mol. Sci. 2019, 20, 854
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Figure 5. Crystallographic diagrams for Sn(SBoVa)Me : (
a
) Molecular structure of the second
) overlay diagram of molecules a (yellow image) and
b (aqua) drawn so the five-membered rings are overlapped and ( ) supramolecular dimer
2
independent molecule, molecule b, (
b
c
.
. .
sustained by edge-to-edge chelate ring benzene interactions (upper image) between Sn1-containing
(chelate ring) .
. .
π (ethoxybenzene) and
molecules, supramolecular layer sustained by edge-to-edge
π
phenyl-C–H . . .
π
(methoxybenzene) interactions occurring between Sn2-containing molecules and a
(chelate ring) .
. .
π
view of the unit cell contents in projection down the b-axis. The edge-to-edge
π
.
. .
(
ethoxybenzene) and C–H π interactions are shown as blue and purple dashed lines, respectively.
In the absence of conventional hydrogen bonding interactions, the crystals of Me Sn(S2MoVa),
2
Me Sn(S4MoVa) and Me Sn(SBoVa) feature a range of weak non-covalent interactions. Packing
2
2
diagrams are given in Figures 3c, 4c and 5c and geometric details characterising the
specified intermolecular interactions are given in Tables S4–S6. In the molecular packing of
.
. .
. . .
Me Sn(S2MoVa), imine-C–H O(phenoxide), methylene-C–H
π
(methoxybenzene), involving
(tolyl) interactions combine to stabilise
supramolecular layers in the ab-plane; layers stack without directional interactions between
them. The crystal of Me Sn(S4MoVa) features additional recognisable points of contact between
2
.
. .
both methylene groups, and tin-bound-methyl-C–H
π
2
.
. .
molecules largely due to the participation of the methoxy substituents. Thus, tolyl-C–H
O
.
. .
(
methoxy) and methoxy-methyl-C–H O (methoxy, phenoxide) interactions are apparent as
are tolyl-C5–H .
. .
π π(tolyl) points of contact. These
(chelate) and tin-bound-methyl-C–H . . .
.
. .
consolidate the three-dimensional architecture, see Figure 4c. The presence of C–H
interactions is of interest and have only relatively recently been appreciated as being important
in contributing to the stability of coordination compounds [58 60]. These interactions usually
stabilise (centrosymmetric) dimeric aggregates [59] and that is the case in Me Sn(S4MoVa), see
π (chelate)
–
2
.
. .
Figure 4c. In the crystal of Me Sn(SBoVa), both phenyl-C–H
π (methoxybenzene) and less common
2
. .
π
(chelate) .
π(methoxybenzene) interactions are formed. The latter interactions have been the
subject of a recent comprehensive review [61]. In the present case, Sn1-containing molecules interact
in this mode via association between the five-membered (Sn/S1/N1/N2/C1) chelate ring and a
methoxybenzene ring. However, these interactions are best described as edge-to-edge interactions
with the closest contact occurring between a nitrogen atom of the chelate ring and the benzene-carbon,
see Figure 5c. Similar edge-to-edge contacts are found for the second independent molecule with

Int. J. Mol. Sci. 2019, 20, 854
12 of 34
the resulting dimeric aggregates being connected into a supramolecular layer in the ab-plane by
phenyl-C–H .
. .
π (methoxybenzene) interactions. Globally, the packing comprises alternating layers of
molecules of a (with no obvious points of contact between the dimeric units) and molecules of b along
the c-axis with no directional interactions between successive layers.
B3LYP-derived bond lengths and angles of Me Sn(S2MoVa), Me Sn(S4MoVa) and Me Sn(SBoVa)
2
2
2
are in excellent agreement with the values obtained from the crystallographic analysis above (see
Table 1), consistent with the absence of strong directional intermolecular interactions in their crystals.
For instance, the deviations in the selected bond lengths/angles of Me Sn(S2MoVa), Me Sn(S4MoVa)
2
2
◦
and Me Sn(SBoVa) ranged from 0.002–0.144 Å/0–5.6 , whereby the maximum bond length deviation
2
corresponded to the Sn–N2 bond for Me Sn(S4MoVa) and the maximum bond angle deviation
2
corresponded to the S1–Sn–O1 angle for Me Sn(SBoVa).
2
2
.6. UV-Vis Absorption Spectroscopy
The experimental UV-vis spectra of the Schiff bases measured in DMSO exhibited prominent
absorption bands at 340–348 and 371–389 nm. The corresponding peaks were predicted at 334 and
77–378 nm using B3LYP. The highest occupied molecular orbital and lowest unoccupied molecular
orbital (HOMO-LUMO) electron density distribution of the Schiff bases and organotin(IV) compounds
see Figure S1) showed that the HOMO was predominantly centred on the thiolate group and
3
(
azomethine moieties of the dithiocarbazate backbone in each of the compounds investigated here.
Conversely, the LUMO was consistently centred on the 2-hydroxy-3-methoxyphenyl ring, as well as on
the backbone of the dithiocarbazate moieties. Thus, the 334 nm absorption peak was attributed to the
n
→π* transitions associated with the non-bonding electron pair of the azomethine nitrogen and sulphur
atoms. There was some π→π* character to this transition as well, relating to electron delocalization
IV
in the aromatic rings. In the spectra of the organotin(IV) compounds, the additional, strong S
→
Sn
IV
intra-ligand band indicated coordination at 444–447 nm. The presence of the S
→
Sn LMCT band
was corroborated by IR evidence which showed that the tin centre was coordinated to the sulphur
atom in the organotin(IV) compounds [62]. The HOMO and LUMO of the organotin(IV) compounds
were comparable to those of the corresponding Schiff bases, i.e., the HOMO was largely centred on
the backbone of the Schiff bases and the LUMO mostly covered the 2-hydroxy-3-methoxyphenyl ring
as well as the backbone of the Schiff base thus explaining the observed n→π* and π→π* transitions
at 307–315 and 353–373 nm, respectively. The HOMO and LUMO transitions were the same for all
the Schiff bases due to their similar structures, the difference being only the presence and position of
the methyl group attached to the phenyl ring. Complete experimental and calculated UV-vis data are
listed in Table S7.
2
.7. Biological Assays
2
.7.1. Cytotoxicity
The Schiff bases and their organotin(IV) compounds were screened for in vitro cytotoxicity against
a panel of 12 cancer cell lines: EJ-28 (muscle invasive bladder), RT-112 (minimum-invasive bladder),
HT29 (colon), U87 and SJ-G2 (glioblastoma), MCF-7 (breast), A2780 (ovarian), H460 (lung), A431 (skin),
DU145 (prostate), BE2-C (neuroblastoma), MIA (pancreatic) and one normal breast cell line (MCF-10A)
using the MTT metabolic assay (Figure 6). After 72 h of incubation, a dose-dependent proliferative
effect towards the cancer cell lines was measured at very low micromolar (µM) concentrations of
the compounds. The growth inhibition concentration of the Schiff bases and their organotin(IV)
compounds required to inhibit 50% cell proliferation relative to the cells that were treated with DMSO
(
control) are given in Table 2. The stability of these compounds was assessed in DMSO as well as in a
mixture of DMSO-H O. The absorbance obtained from UV-vis spectroscopy analysis was monitored
2
for 72 h, and an unchanged pattern in the spectra was indicative that the compounds were stable in
both the solvent systems tested.

Int. J. Mol. Sci. 2019, 20, 854
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As outlined earlier, the effect of the substitution in the methyl group on the dithiocarbazate
backbone on the coordination with the tin ion was explored. The GI50 values for the three Schiff bases
showed a slight difference; the presence of methyl group at ortho (S2MoVaH) and para (S4MoVaH)
positions resulted in a slight reduction in potency as compared to SBoVaH. This cytotoxicity difference
was probably due to the hydrophobic nature of the methyl group [63]. To note, S4MoVaH exhibited
the lowest potency against HT29 and MIA with GI50 values of 8.0
It was also observed that the coordination of the Schiff bases with diphenyltin(IV) induced potent
selective growth inhibition in A2780, BE2-C and MIA cells, in which the compounds exhibited potency
±
3.5 and 11
±
2.3 µM, respectively.
higher than their respective Schiff bases. Cytotoxic selectivity was also observed for Ph Sn(SBoVa)
2
and Ph Sn(S4MoVa) against MCF-7 with GI values of 0.90
±
0.16 and 0.82
This constituted a potency that was almost thrice higher than that of the corresponding Schiff bases,
SBoVaH and S4MoVaH which had values of 2.5 0.12 and 3.5 0.00 M, respectively. In addition,
0.22 M) than S4MoVaH
M) against HT-29 cancer cells. A 3-fold greater activity against SJ-G2 was
0.15 and 0.84 0.09
±
0.14 µM, respectively.
2
50
±
±
µ
Ph Sn(S4MoVa) exhibited a much higher potency (GI value of 0.87
±
µ
2
50
(
GI50 value of 8.0
±
3.5 µ
observed for Ph Sn(SBoVa) and Ph Sn(S2MoVa) with GI values of 0.74
±
±
µM
2
2
50
respectively, than their respective Schiff bases. It is noteworthy to mention that the diphenyltin(IV)
compounds showed high selectivity against EJ-28, RT-112, MCF-7, A2780, BE2-C, SJ-G2 and MIA cell
lines in the range 1.4-5.0-fold as compared to the normal breast cell line (MCF10A).
By contrast, dimethyltin(IV) compounds showed almost similar cytotoxicity values when assayed
against the panel of cancer cell lines suggesting that the presence of two phenyl groups coordinated
directly to the tin ion improved the potency of the Schiff bases. Electron delocalisation over the
chelate rings, which increased the lipophilicity of the molecules and favoured their permeation
through the lipid layer of the cancer cell membranes could also be a contributing factor to the
enhancement of cytotoxicity [64]. A third contributing factor could be the ease of ability of the
phenyl planes to intercalate into the base pairs of DNA in the cancer cell lines [8]. To better understand
the mode of biological action of the compounds that showed promising activity, apoptosis assays
using the minimally invasive bladder cancer RT-112 cells and DNA binding studies were carried out
by evaluating the compounds’ mechanism of action.
2
.7.2. Annexin V Assays of Ph Sn(S2MoVa)Ph , Ph Sn(S4MoVa), and Ph Sn(SBoVa)-Treated
2 2 2 2
RT-112 Cells
Cell death usually occurs by apoptosis or necrosis, the latter induces severe inflammation.
The apoptotic cells containing apoptotic bodies (small membrane-bound vesicles), are engulfed by
macrophages, and hence no inflammatory response is triggered. On this account, as apoptotic cell
induction is considered crucial in cytotoxic drug development, investigations using the Annexin V
assay were performed to study the apoptosis effect on cancer cells of the organotin(IV) compounds.
The three most highly cytotoxic diphenyltin(IV) compounds at their specific IC50 concentrations
were chosen for this assay. The analysis was carried out using RT-112, minimum-invasive bladder
cancer cells with high levels of epidermal growth factor receptor expression. In the early stages of
apoptosis, changes occurred at the cell surface [65]. The plasma membrane alteration occurs due to
the translocation of phosphatidyl-serine (PS) from the inner part of the plasma membrane where it is
normally located on the cytoplasmic surface, to the outer layer. The PS exposed on the cell surface
has high binding affinity to the Annexin V. In the later stages of apoptosis, as well as necrosis, the cell
membranes lose their membrane integrity and allows propidium iodide (PI) to enter into the nucleus
−
−
of the cells. Annexin V/PI double staining was used to differentiate viable cells (Annexin V and PI )
+
−
+
+
in early apoptosis (Annexin V and PI ) or late apoptosis (Annexin V and PI ) and necrosis (annexin
−
+
V
and PI ) stages [66]. Under fluorescence microscopy, the cells stained by annexin V appear green,
whereas the cells stained by PI appear red. After 24 h exposure to the diphenyltin(IV) compounds,
Figure 6 show that the cells undergoing late apoptosis and necrosis features. Thus, this assay proved
that the diphenyltin(IV) compounds trigger the activation of apoptosis [67].

Int. J. Mol. Sci. 2019, 20, 854
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Table 2. Summary of the in vitro cytotoxicity of the Schiff bases and their organotin(IV) compounds in several cell lines, determined by MTT assay and expressed as
GI50 values with standard errors. (GI50 is the concentration at which cell growth is inhibited by 50% over 72 h).
Growth Inhibition Concentration, GI50 (µM)
Compounds
EJ-28
RT-112
4.6 ± 0.45
0.31 ± 0.05
2.4 ± 0.77
3.2 ± 0.51
1.7 ± 0.49
>5.0
3.7 ± 0.57
0.58 ± 0.29
2.4 ± 0.33
3.56 ± 0.88
HT29
U87
MCF-7
2.7 ± 0.21
1.4 ± 0.12
3.1 ± 0.12
3.5 ± 0.00
0.82 ± 0.14
3.3 ± 0.32
2.5 ± 0.12
0.90 ± 0.16
2.5 ± 0.21
6.5 ± 0.8
A2780
H460
A431
Du145
BE2-C
SJ-G2
MIA
MCF10A
3.1 ± 0.07
1.2 ± 0.30
3.0 ± 0.09
3.5 ± 0.23
1.1 ± 0.37
3.3 ± 0.15
3.0 ± 0.07
1.3 ± 0.20
2.8 ± 0.41
nd
S2MoVaH
Ph2Sn(S2MoVa)
Me2Sn(S2MoVa)
S4MoVaH
Ph2Sn(S4MoVa)
Me2Sn(S4MoVa)
SBoVaH
Ph2Sn(SBoVa)
Me2Sn(SBoVa)
Cisplatin
3.4 ± 0.47
0.35 ± 0.05
2.5 ± 0.78
3.6 ± 0.44
3.1 ± 0.37
>5.0
3.1 ± 0.20
0.32 ± 0.05
3.4 ± 0.30
3.97 ± 0.48
3.9 ± 0.71
1.8 ± 0.13
2.7 ± 0.10
8.0 ± 3.5
4.3 ± 0.30
4.4 ± 1.7
3.6 ± 0.23
3.9 ± 0.00
1.3 ± 0.40
3.3 ± 0.18
3.1 ± 0.26
1.1 ± 0.39
4.5 ± 0.19
4.0 ± 1.0
3.2 ± 0.15
0.23 ± 0.01
3.1 ± 0.06
3.1 ± 0.40
0.35 ± 0.07
3.1 ± 0.63
3.0 ± 0.06
0.23 ± 0.03
3.2 ± 0.03
1.0 ± 0.1
4.3 ± 0.12
2.8 ± 0.00
3.3 ± 0.13
5.5 ± 0.73
1.9 ± 0.23
4.7 ± 0.46
3.3 ± 0.21
2.2 ± 0.00
3.8 ± 0.10
0.9 ± 0.2
3.8 ± 0.03
2.1 ± 0.09
3.0 ± 0.03
4.2 ± 0.74
1.4 ± 0.38
4.2 ± 0.34
3.0 ± 0.15
1.6 ± 0.07
3.2 ± 0.03
2.4 ± 0.3
4.1 ± 0.09
1.7 ± 0.07
3.9 ± 0.03
5.4 ± 0.23
1.5 ± 0.31
4.2 ± 0.10
3.4 ± 0.30
1.4 ± 0.33
3.2 ± 0.13
1.2 ± 0.1
3.1 ± 0.00
0.47 ± 0.05
2.9 ± 0.03
3.6 ± 0.27
0.53 ± 0.01
3.3 ± 0.23
2.8 ± 0.20
0.50 ± 0.05
2.7 ± 0.13
1.9 ± 0.2
3.0 ± 0.09
0.84 ± 0.09
2.5 ± 0.30
3.3 ± 0.87
1.0 ± 0.16
3.1 ± 0.55
2.4 ± 0.26
0.74 ± 0.15
2.1 ± 0.12
0.4 ± 0.1
4.9 ± 0.37
0.34 ± 0.02
3.9 ± 0.07
11 ± 2.3
0.87 ± 0.22
6.9 ± 3.5
0.39 ± 0.03
8.9 ± 1.30
4.5 ± 0.25
0.43 ± 0.04
5.0 ± 0.09
8.0 ± 1.0
2.2 ± 0.033
1.2 ± 0.22
2.8 ± 0.15
11.0 ± 2.0
GI50 (µM): (the colors indicate) = 0.1–0.99 ; = 1.0–9.9 ; = 10–100 ; = nd (not determined) .

Int. J. Mol. Sci. 2019, 20, 854
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2
.7.3. Measurement of Reactive Oxygen Species (ROS) in RT-112 Cells Treated with Ph Sn(S2MoVa)
2
and Ph Sn(S4MoVa)
2
Two compounds with the most promising bioactivity, Ph Sn(S2MoVa) and Ph Sn(S4MoVa), were
2
2
selected for further cell apoptosis studies, in order to evaluate the primary damage to biological
macromolecules through redox reactions, particularly via formation of ROS [68]. ROS are highly
•
•
reactive molecules/radicals (O , O , H O , HO ) that are continuously generated during aerobic
2
2
2
2
metabolism, but when in excessive amounts, can lead to protein and DNA oxidation, protein
cross-linking and cell death. Estimation of the levels of ROS in cell culture is an important step
towards better understanding the mechanisms that lead to cell death or disease processes. Figure 7
shows that both the Ph Sn(S2MoVa) and Ph Sn(S4MoVa) stimulated ROS generation. ROS production
2
2
was measured using the ROS-detecting fluorescent dye DCFH-DA and DMSO was used as a control.
Both compounds were able to induce ROS production in RT-112 cell lines after 24 h. The significant
increase in ROS generation supports the findings that the compounds have high potential cytotoxicity
and suggested that the mechanism of cell death was via the simulation of mitochondrial-initiated
events [69].
Figure 6. Apoptosis detection through fluorescence microscopy. Cells were treated for 24 h with
Ph Sn(SBoVa) (0.58 µM), Ph Sn(S2MoVa) (0.31 µM) and Ph Sn(S4MoVa) (1.66 µM) and the negative
2 2
2
control (DMSO) in complete media. After staining with Annexin V and PI, necrotic and apoptotic cells
were detected by fluorescence microscopy (20×).

Int. J. Mol. Sci. 2019, 20, 854
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Figure 7. Percent reactive oxygen species (ROS) production in RT-112 cells treated with
(
a
) Ph Sn(S2MoVa) (0.31
µ
M) (
b
) Ph Sn(S4MoVa) (1.66
µ
M) for 24 h and stained with 1 mM DCFH-DA
2
2
◦
for 60 minutes at 37 C. DMSO and H O acted as negative and positive controls, respectively.
2
2
2
.7.4. DNA Interaction Studies
Both theoretical and experimental DNA binding of the synthesised organotin(IV) compounds
with DNA were investigated. The interaction of organotin(IV) compounds with CT-DNA was
elucidated via UV-visible absorption spectroscopy. The electronic spectra of the organotin(IV)
compounds exhibited three absorption bands around 306–315 nm, 350–377 nm and 433–450 nm,
which corresponded to intra-ligand transitions and ligand metal charge transfer (LMCT) transitions
(
(
see below). The absorption spectra of diphenyltin(IV) compounds at a constant concentration
50 M) in the absence and the presence of different concentrations of CT-DNA are shown in Figure 8.
µ
The DNA binding spectra for dimethyltin(IV) compounds are presented in Figure S2. Upon increasing
concentrations of CT-DNA, hypochromism and bathochromism occurred at around 350–377 nm
indicating strong
compounds and the nitrogeneous pair of the DNA strand [11
bathochromism shifts were also observed in other transitions. In order to compare the binding strength
of the organotin(IV) compounds to CT-DNA, the intrinsic binding constants (K ) were quantitatively
π–π
stacking interactions between the aromatic chromophore of the organotin(IV)
,70]. The same hypochromism and
b
determined and are presented in Table 3. The K data showed that the compounds had strong binding
b
affinities, potentially enabling them to block enzyme bound to the nitrogenous bases of DNA.
The Gibbs free energies of interaction between the organotin(IV) compounds and DNA ranged
−
1
from
−
31.0 to
−
37.6 kJ mol , meaning that the interaction of the organotin(IV) compounds with
DNA was spontaneous [71]. To investigate and understand the interactions between DNA and these
compounds, molecular docking simulations were carried out.
Table 3. Binding constants (K ), hypochromism (%), bathochromic shifts (nm) and Gibbs free energy
b
(
kJ mol 1) values for the interaction of organotin(IV) compounds with calf thymus DNA (CT-DNA).
−
Binding Constant
K )
Hypochromism,
(%)
Bathochromism Shift
(nm)
Gibbs Free Energy
Compounds
−
1
(
(kJ mol
)
b
5
5
5
5
6
6
Ph Sn(S2MoVa)
6.21 × 10
3.46 × 10
2.67 × 10
4.15 × 10
1.21 × 10
3.91 × 10
41.06
37.97
28.25
43.72
70.72
53.26
6
10
4
7
12
1
−33.1
−31.6
−31.0
−32.1
−34.7
−37.6
2
Me Sn(S2MoVa)
2
Ph Sn(S4MoVa)
2
Me Sn(S4MoVa)
2
Ph Sn(SBoVa)
2
Me Sn(SBoVa)
2

Int. J. Mol. Sci. 2019, 20, 854
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Figure 8.
(
a
) Electronic absorption spectra of (
iii) Ph Sn(SBoVa); (
) Plot of [DNA]/εa − εf vs. [DNA] for absorption titration of DNA with
) Ph Sn(S2MoVa), (ii) Ph Sn(S4MoVa) and (iii) Ph Sn(SBoVa). The arrow indicates the change
i) Ph Sn(S2MoVa), (ii) Ph Sn(S4MoVa) and
2 2
(
(
b
2
i
2
2
2
in absorbance in tandem with increasing DNA concentration.
2
.8. Molecular Docking Studies
Molecular docking analyses of the organotin(IV) compounds with the DNA duplex of sequence
d(CGCGAATTCGCG)2 dodecamer (PDB ID: 1BNA) were performed and results are detailed in
Table 4. These simulations demonstrated that the organotin(IV) compounds considered here were
commensurate with the dimensions of the grooves of the DNA duplex sequence. Figure 9a revealed

Int. J. Mol. Sci. 2019, 20, 854
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that these small organotin(IV) compounds fitted well into the A-T rich region of the minor grooves of
DNA. Generally, the electronegativity of A-T sequences provided better van der Waals interactions
due to their narrower sequences compared to G-C regions. In groove regions, van der Waals and
hydrophobic interactions play important roles in the stabilisation of compounds [72]. Their interactions
therefore, involved hydrogen bonding, hydrophobic and electrostatic interactions with lowest binding
−
1
−1
energies between
−
9.10 to
−
10.07 kcal mol , which is better than cisplatin ( 8.65 kcal mol ) [25].
−
The interactions occurred either through the phosphate-oxygen atom of the DNA or via DNA bases.
Ph Sn(S2MoVa) exhibited two hydrogen bonding interactions through methoxy-oxygen atoms and
2
methyl groups with phosphate-oxygen atoms of DT20 and DT8, respectively. Similar interactions
were observed for Me Sn(S2MoVa) through the oxygen atom of methoxy group with the phosphate
2
backbone atoms of DT19; Ph Sn(S4MoVa), Me Sn(S4MoVa) and Me Sn(SBoVa) interacted through
2
2
2
the carbon atoms of the azomethine group with oxygen atoms of thymine base, DT8, DT7 and DT20,
respectively. Detailed interactions between the organotin(IV) compounds and DNA residues are
presented in Figure 9b. Thus, on the basis of docking studies, it could be inferred that the cytotoxic
activities of organotin(IV) compounds could have arisen due to effective interactions of the compounds
with DNA.

Int. J. Mol. Sci. 2019, 20, 854
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Table 4. Molecular docking data for the organotin(IV) compounds with B-DNA (PDB ID: 1BNA) dodecamer d(CGCGAATTCGCG)2.
−
1
Final Intermolecular Energy, kcal mol
Final Total Internal
Energy (2),
Torsional Free
Energy (3),
kcal mol
Unbound System’s
Energy [=(2)] (4),
Estimated Free Energy of
Estimated Free
Energy of Binding,
Complex
Binding [(1) + (2) + (3) − (4)],
vdW + Hbond +
desolv. Energy
Electrostatic
Energy
Total
(1)
−
1
−1
−1
−1
−1
kJ mol
kcal mol
kcal mol
kcal mol
Ph Sn(S2MoVa)
−11.46
−10.27
−11.87
−10.84
−11.07
−10.58
0.00
−0.03
0.01
0.00
0.03
−11.47
−10.30
−11.86
−10.83
−11.04
−10.59
−2.19
0.57
−1.82
−0.53
−1.86
−0.54
1.79
1.19
1.79
1.19
1.79
1.19
−2.19
−0.57
−1.82
−0.53
−1.86
−0.54
−9.68
−9.10
−40.50
−38.07
−42.13
−40.33
−38.70
−39.33
2
Me Sn(S2MoVa)
2
Ph Sn(S4MoVa)
−10.07
−9.64
−9.25
−9.40
2
Me Sn(S4MoVa)
2
Ph Sn(SBoVa)
2
Me Sn(SBoVa)
−0.01
2

Int. J. Mol. Sci. 2019, 20, 854
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Figure 9. (a) Schematic representation of organotin(IV) compounds that fit well in the grooves of
the DNA strand obtained by docking simulations. The two double-stranded DNA comprise of the
phosphate deoxyribose backbone with guanine (DG, red), cytosine (DC, blue), adenine (DA, pink) and
thymine (DT, orange). (b) Molecular interactions of organotin(IV) compounds within the grooves of
double stranded DNA residues.

Int. J. Mol. Sci. 2019, 20, 854
21 of 34
3
. Experimental
3
.1. Physical Measurements
Melting points were determined using an Electrothermal digital melting point apparatus
Cole-Parmer, Staffordshire, UK). IR spectra were recorded using the Perkin Elmer Spectrum 100
(
−
1
with Universal ATR Polarization (PerkinElmer, Boston, MA, USA) in the range 4000–280 cm . C,
H, and N elemental analyses were carried out using a LECO CHNS-932 instrument (LECO, Saint
−
3
Joseph, MI, USA). Molar conductivities of 10 M solutions of the organotin(IV) compounds in DMSO
◦
were measured at 27 C using a Jenway 4310 conductivity meter fitted with a dip-type cell with a
platinised electrode (Cole-Parmer, Staffordshire, UK). Electronic spectra were recorded on a Shimadzu
UV-1650 PC recording spectrophotometer (1000–200 nm) (Shimadzu, Tokyo, Japan). 1H and
13
C
NMR spectra were recorded using an NMR JNM ECA400 spectrometer (JEOL, Peabody, MA, USA)
with tetramethylsilane (TMS) was used as an internal standard. 119Sn NMR were recorded using a
JOEL NMR spectrophotometer (JEOL, Peabody, MA, USA). The mass spectra were recorded using a
Shimadzu GC-MS QP2010Plus mass spectrometer (Shimadzu, Tokyo, Japan).
3
.2. Materials
All solvents and reagents were of analytical reagent grade and used without further purification.
Chemicals: Hydrazine hydrate, 80% (Fluka, Buchs, Switzerland), benzylchloride, 99% (Merck,
≥
New York, NY, USA), 2-methybenzyl chloride, 99% (ACROS, Carson, CA, USA), 4-methylbenzyl
chloride (ACROS, Carson, CA, USA), potassium hydroxide (HmbG, Hamburg, Germany), carbon
disulfide (BDH, Radnor, PA, USA), 2-hydroxy-3-methoxybenzaldehyde (Merck, New York, NY, USA)
and nitric acid, 65% (Thermo Fisher Scientific Inc., Waltham, MA, USA), dichlorodiphenyltin(IV)
and dichlorodimethyltin(IV) (Sigma Aldrich, St. Louis, MO, USA). Solvents: Acetonitrile (Baker,
Sanford, ME, USA), absolute ethanol, 99.8% (Scharlau, Barcelona, Spain), ethanol, 95% (John Kollin
Corporation, Midlothian, UK), methanol (Fisher, Pittsburgh, PA, USA) and dimethylsulfoxide
(Scharlau, Barcelona, Spain).
Chemicals used for biological assay were purchased as follows: RPMI-1640 medium with
L-glutamine (without sodium bicarbonate) (Sigma Aldrich, St. Louis, MO, USA), Penicillin/Streptomycin
solution (Biowest, Riverside, MO, USA), Fetal Bovine Serum (sterile filtered) (Biowest, Riverside, MO,
USA), Trypsin 0.25%, EDTA in HSSS without calcium, magnesium, with phenol red (Biowest, Riverside,
MO, USA), Phosphate Buffered Saline (PBS) 10
dimethyl sulfoxide (DMSO) (Fisher Scientific, Pittsburgh, PA, USA), Annexin-V-FLUOS Staining
Kit containing propidium iodide (PI), Annexin-V-Fluorescein, and incubation buffer (ROCHE, Basel,
×
concentrate (Sigma Aldrich, St. Louis, MO, USA),
Switzerland). OxiSelect
,7-dichlorofluorescein (DCF) Standard, Hydrogen Peroxide, and 2
San Diego, CA, USA).
™
Intracellular ROS Assay Kit (Green Fluorescence) containing 20
×
DCFH-DA,
2
×
Cell Lysis Buffer (Cell Biolabs,
3
.3. Synthesis
3
.3.1. S-N-R-Benzyldithiocarbazates (N = 2,3, R = Methyl)
The procedure was adapted from that reported by Tarafder and co-workers [73
–75]. Potassium
hydroxide (11.4 g, 0.2 mol) was dissolved in ethanol (70 mL, 90%). Then, hydrazine hydrate (10 g,
◦
0
.2 mol) was added and the mixture was maintained at 0 C in an ice-salt bath. Carbon disulfide
(
15.2 g, 0.2 mol) was added dropwise with vigorous stirring (750 rpm) over a period of 1 h. The two
layers that formed were separated and the light-brown lower layer was dissolved in 40% ethanol
◦
(
60 mL) below 5 C. The mixture was kept in an ice-bath and 2-methylbenzyl chloride/4-methylbenzyl
chloride/benzyl chloride (0.2 mol) was added dropwise with vigorous stirring. The sticky white
product, which formed, S2MBDTC, S4MBDTC or SBDTC, was filtered and left to dry overnight in a
desiccator over anhydrous silica gel. The products were then kept in a freezer.

Int. J. Mol. Sci. 2019, 20, 854
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3
.3.2. S-2-Methybenzyl-β-N-(2-hydroxy-3-methoxybenzylmethylene) Dithiocarbazate (S2MoVaH)
The synthesis of the Schiff base, S2MoVaH was adapted from the literature [76 77].
,
S-2-methylbenzyldithiocarbazate (S2MBDTC) (2.12 g, 10 mmol) was dissolved in hot acetonitrile
3
(
100 cm ) and added to an equimolar amount of 2-hydroxy-3-methoxybenzaldehyde (1.52 g, 10 mmol)
3
◦
in absolute ethanol (20 cm ). The mixture was heated (80 C) with continuous stirring for about 30 min
and then allowed to stand overnight at room temperature. The resultant product was recrystallised
from CH CN/EtOH (1:1) to give a light yellow crystalline solid that was filtered and washed with
3
◦
cold absolute ethanol. Yield: 65%. M.p.: 180–183 C. Elemental analysis: calculated for C H N O S :
C, 58.93; H, 5.24; N, 8.09. Found: C, 59.75; H, 5.25; N, 7.81. FT-IR (ATR, cm ): 3084, (N–H); 1600,
C=N); 1117, (N–N); 1026, (C=S). H NMR (DMSO-d )
1
7
18
2
2 2
−
1
1
(
δ
(ppm.): 13.34 (s,1H, NH), 9.57 (s, 1H, OH),
6
8
.51 (s, 1H, CH), 6.75–7.34 (multiplet, 7H, Ar–H), 4.40 (s, 2H, CH ), 3.76 (s, 3H, O–CH ), 2.30 (s, 3H,
2 3
13
Bz–CH3); C NMR (DMSO-d6)
δ (ppm.): 196.1 (C=S), 148.6 (C=N); 148.6, 147.4, 144.9, 137.4, 134.4,
1
30.8, 130.7, 128.2, 126.7, 120.0, 118.8, 114.4 (aromatic-C), 56.4 (O–CH ), 36.9 (CH ), 19.4 (CH ). m/z
3
2
3
calculated for C H N O S : 346.47, found: 346.15.
17
18
2
2 2
3
.3.3. S-4-Methybenzyl-β-N-(2-hydroxy-3-methoxybenzylmethylene)dithiocarbazate (S4MoVaH)
The synthesis of the Schiff base, S4MoVaH was similar to S2MoVaH [76 77].
,
S-4-
3
methylbenzyldithiocarbazate (S4MBDTC) (2.12 g, 10 mmol) was dissolved in hot acetonitrile (100 cm )
and added to an equimolar amount of 2-hydroxy-3-methoxybenzaldehyde (1.52 g, 10 mmol) in absolute
3
◦
ethanol (20 cm ). The mixture was heated (80 C) with continuous stirring for about 30 min and then
allowed to stand overnight at room temperature. The resultant product was recrystallised from
CH CN/EtOH (1:1) to give a light yellow crystalline solid that was filtered and washed with cold
3
◦
absolute ethanol. Yield: 68%. M.p.: 177–178 C. Elemental analysis: Calculated for C H N O S : C,
8.93; H, 5.24; N, 8.09. Found: C, 58.48; H, 5.18; N, 8.76. FT-IR (ATR, cm ): 3092, (N–H); 1598, (C=N);
118, (N–N); 1030, (C=S). H NMR (DMSO-d )
CH), 6.97–7.24 (multiplet, 7H, Ar–H), 4.39 (s, 2H, CH ), 3.76 (s, 3H, O–CH ), 2.23 (s,3H, Bz–CH );
1
7
18
2
2 2
−
1
5
1
1
δ
(ppm.): 13.32 (s,1H, NH), 9.61 (s, 1H, OH), 8.51 (s, 1H,
6
1
3
C
2
3
3
NMR (DMSO-d6)
δ
(ppm.): 196.2 (C=S), 148.6 (C=N); 148.6, 147.4, 145.0, 137.0, 134.0, 129.7, 129.6, 120.0,
1
3
18.8, 114.4 (aromatic-C), 56.4 (O–CH ), 38.0 (CH ), 21.2 (CH ). m/z calculated for C H N O S :
46.47, found: 346.10.
3
2
3
17 18
2
2 2
3
.3.4. S-Benzyl-β-N-(2-hydroxy-3-methoxybenzylmethylene) Dithiocarbazate (SBoVaH)
The synthesis of Schiff base, SBoVaH was adapted from literature [76
SBDTC) (1.98 g, 10 mmol) was dissolved in hot absolute ethanol (100 cm ) and added to an
,
77] S-benzyldithiocarbazate
3
(
3
equimolar amount of 2-hydroxy-3-methoxybenzaldehyde (1.52 g, 10 mmol) in absolute ethanol (20 cm ).
◦
The mixture was heated (78 C) with continuous stirring for about 30 min and then allowed to stand
overnight at room temperature. The resultant product was recrystallised from ethanol to yield light
◦
yellow crystals that were filtered and washed with cold absolute ethanol. Yield: 70%. M.p.: 173–174 C.
Elemental analysis: calculated for C H N O S : C, 57.81; H, 4.85; N, 8.43. Found: C, 58.21; H, 4.87;
1
6
16
2
2 2
−
1
1
N, 8.28. FT-IR (ATR, cm ): 3089, (N–H); 1598, (C=N); 1125, (N–N); 1030, (C=S). H NMR (DMSO-d )
6
δ
(ppm.): 13.34 (s,1H, NH), 9.58 (s, 1H, OH), 8.52 (s, 1H, CH), 6.77-7.37 (multiplet, 8H, Ar–H), 4.45 (s,
H, CH ), 3.77 (s, 3H, O–CH ); C NMR (DMSO-d6) δ (ppm.): 196.1 (C=S), 148.6 (C=N); 148.6, 147.4,
2 3
13
2
1
44.9, 137.3, 129.8, 129.0, 127.8, 120.0, 118.8, 114.4 (aromatic-C), 56.4 (CH ), 38.1 (CH ). m/z calculated
3 2
for C H N O S : 332.44, found: 332.10.
16
16
2
2 2
3
.3.5. Diphenyltin(IV) Compounds
3
Each Schiff base (1 mmol) was dissolved in absolute ethanol (50 cm ) and mixed with an ethanolic
3
solution (10 cm ) of Ph SnCl (0.34 g, 1 mmol). The resultant yellow solution was refluxed for ca. 6 h.
2
2
The mixture was left overnight at room temperature and the reduction in the volume of the reaction

Int. J. Mol. Sci. 2019, 20, 854
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mixture resulted in the deposition of a yellow precipitate which was filtered off and recrystallised
from methanol.
Diphenyltin(IV) [S-2-Methybenzyl-β-N-(2-hydroxy-3-methoxybenzylmethylene) dithiocarbazate],
Ph Sn(S2MoVa)
2
◦
Yellow solid. Yield: 44%. Melting point: 149–150 C. Elemental analysis: Calculated for
−
1
C H N O S Sn: C, 56.42; H, 4.24; N, 4.54. Found: C, 56.52; H, 4.97; N, 4.06. FT-IR (ATR, cm ): 1588,
C=N); 1076, (N–N); 963, (C=S). H NMR (CDCl ) δ (ppm.): 8.77 (s, 1H, CH), 4.47 (s, 2H, CH ), 2.43 (s,
3 2
H, CH ), 3.97 (s, 3H, O–CH ), 6.69-7.94 (multiplet, 17H, Ar–H); C NMR (CDCl3)
29
34
2
6 2
1
(
3
13
δ
(ppm.): 171.9
3
3
(
(
C–S), 166.1 (C=N); 159.3, 152.0, 142.0, 136.1, 130.6, 130.4, 130.2, 128.9, 127.9, 126.3, 126.1, 117.2, 116.2
119
aromatic-C), 56.6 (O–CH ), 34.4 (CH ), 19.4 (Bz–CH ). Sn NMR (CDCl ) δ (ppm.): −236.38.
3
2
3
3
Diphenyltin(IV) [S-4-Methybenzyl-β-N-(2-hydroxy-3-methoxybenzylmethylene) dithiocarbazate],
Ph Sn(S4MoVa)
2
◦
Yellow solid. Yield: 72%. Melting point: 130–132 C. Elemental analysis calculated for
−
1
C H N O S : C, 54.82; H, 4.44; N, 4.41. Found: C, 54.42; H, 4.05; N, 4.18. FT-IR (ATR, cm ):
589, (C=N); 1068, (N–N); 958, (C=S). H NMR (CDCl ) δ (ppm.): 8.74 (s, 1H, CH), 4.41 (s, 2H, CH ),
3 2
.32 (s, 3H, CH ), 3.94 (s,3H, O–CH ), 6.69-7.93 (multiplet, 17H, Ar–H); C NMR (CDCl3)
17
17
2
2 2
1
1
2
13
δ
(ppm.):
3
3
1
1
71.8 (C–S), 166.1 (C=N), 159.3, 152.0, 142.0, 137.2, 136.0, 135.8, 133.5, 130.2, 129.4, 129.2, 128.9, 126.1,
119
17.2, 116.3 (aromatic-C), 56.6 (O–CH ), 36.0 (CH ), 21.2 (Bz–CH ). Sn NMR (CDCl3) δ (ppm.):
3 2 3
−
236.22.
Diphenyltin(IV) [S-Benzyl-
β
-N-(2-hydroxy-3-methoxybenzylmethylene) dithiocarbazate], Ph Sn(SBoVa)
2
◦
Yellow crystals. Yield: 51%. Melting point: 131–133 C. Elemental analysis calculated for
−
1
C H N O S Sn: C, 55.74; H, 4.01; N, 4.64. Found: C, 56.35; H, 3.91; N, 4.98. FT-IR (ATR, cm ): 1579,
C=N); 1019, (N–N); 958, (C=S). H NMR (CDCl ) δ (ppm.): 8.74 (s, 1H, CH), 4.45 (s, 2H, CH ), 3.96 (s,
3 2
H, O-CH ), 6.69-7.93 (multiplet, 18H, Ar–H); C NMR (CDCl3) δ (ppm.): 171.6 (C–S), 166.2 (C=N),
3
28
24
2
2 2
1
(
3
13
1
5
52.0, 141.9, 136.7, 136.1, 130.3, 129.3, 128.9, 128.7, 127.5, 126.1, 117.2, 117.1, 116.2, 115.2, (aromatic-C),
119
6.6 (O–CH ), 36.1 (CH ). Sn NMR (CDCl ) δ (ppm.): −236.07.
3
2
3
3
.3.6. Dimethyltin(IV) Compounds
3
Each Schiff base (1 mmol) was dissolved separately in absolute ethanol (50 cm ) and
3
3
dichloromethane (DCM) (20 cm ) and then mixed with an ethanolic solution (10 cm ) of Me SnCl
2
2
(
0.22 g, 1 mmol). The resultant yellow solution was refluxed for ca. 6 h. The mixture was left overnight
at room temperature and kept for few days yielding brown crystals that were suitable for single crystal
X-ray diffraction analysis.
Dimethyltin(IV) [S-2-Methybenzyl-β-N-(2-hydroxy-3-methoxybenzylmethylene) dithiocarbazate],
Me Sn(S2MoVa)
2
◦
Yellow crystals. Yield: 78%. Melting point: 128–130 C. Elemental analysis calculated for
−
1
C H N O S Sn: C, 46.27; H, 4.50; N, 5.68. Found: C, 46.52; H, 4.51; N, 5.82. FT-IR (ATR, cm ): 1580,
19
22
2
2 2
1
(
(
C=N); 1076, (N–N); 959, (C=S). H NMR (CDCl ) δ (ppm.): 8.76 (s, 1H, CH), 4.42 (s, 2H, CH ), 2.42
3
2
13
s, 3H, CH ), 3.85 (s,3H, O–CH ), 6.69–7.34 (multiplet, 7H, Ar–H), 0.97 (Sn–CH ); C NMR (CDCl3) δ
3 3 3
(
1
ppm.): 173.9 (C–S), 166.3 (C=N), 158.5, 151.5, 137.2, 134.1, 130.6, 130.4, 127.9, 126.3, 126.1, 116.9, 116.5,
119
15.9 (aromatic-C), 56.3 (CH ), 34.6 (CH ), 19.4 (Bz–CH ), 7.1 (Sn–CH ). Sn NMR (CDCl3)
δ
(ppm.):
3
2
3
3
−
109.41.
Dimethyltin(IV) [S-4-Methybenzyl-β-N-(2-hydroxy-3-methoxybenzylmethylene) dithiocarbazate],
Me Sn(S4MoVa)
2
◦
Yellow crystals. Yield: 55%. Melting point: 98–102 C. Elemental analysis calculated for
−
1
C H N O S Sn: C, 46.27; H, 4.50; N, 5.68. Found: C, 47.07; H, 4.66; N, 6.11. FT-IR (ATR, cm ):
19
22
2
2 2

Int. J. Mol. Sci. 2019, 20, 854
24 of 34
1
1
2
589, (C=N); 1071, (N–N); 958, (C=S). H NMR (CDCl ) δ (ppm.): 8.73 (s, 1H, CH), 4.36 (s, 2H, CH ),
3 2
13
.32 (s, 3H, CH ), 3.84 (s, 3H, O-CH ), 6.69–7.27 (multiplet, 7H, Ar–H), 0.95 (Sn–CH ); C NMR
3
3
3
(
1
CDCl3) δ (ppm.): 173.7 (C–S), 166.2 (C=N), 158.5, 151.5, 137.1, 133.6, 129.2, 126.1, 119.5, 116.8, 116.4,
119
15.9 (aromatic-C), 56.3 (CH ), 36.2 (CH ), 21.2 (Bz–CH ), 7.1 (Sn–CH ).
Sn NMR (CDCl3) δ (ppm.):
3
2
3
3
−
109.17.
Dimethyltin(IV) [S-Benzyl-
β
-N-(2-hydroxy-3-methoxybenzylmethylene) dithiocarbazate], Me Sn(SBoVa)
2
◦
Yellow crystals. Yield: 48%. Melting point: 104–106 C. Analysis calculated for C H N O S Sn:
18
20
2
2 2
−
1
C, 45.11; H, 4.21; N, 5.85. Found: C, 45.36; H, 4.16; N, 6.02. FT-IR (ATR, cm ): 1581, (C=N); 1026,
(ppm.): 8.73 (s, 1H, CH), 4.40 (s, 2H, CH ), 3.85 (s, 3H, O–CH ),
.69-7.40 (multiplet, 8H, Ar–H), 0.95 (Sn–CH3); C NMR (CDCl3) δ (ppm.): 173.6 (C–S), 166.3 (C=N),
1
(
6
N–N); 959, (C=S). H NMR (CDCl )
δ
3
2
3
13
1
7
58.5, 151.5, 136.8, 129.3, 128.7, 127.4, 126.1, 116.9, 116.5, 115.9 (aromatic-C), 56.3 (O–CH ), 36.4 (CH ),
3
2
.1 (Sn–CH3); 1 Sn NMR (CDCl ) δ (ppm.): −108.94.
19
3
3
.4. Single Crystal X-ray Structure Determination
Crystals suitable for single-crystal X-ray diffraction studies were obtained for Me Sn(S2MoVa),
2
Me Sn(S4MoVa) and Me Sn(SBoVa). Unfortunately, suitable crystals for the diphenyltin(IV)
2
2
compounds were not able to be obtained despite repeated crystal-growth attempts using various
techniques and solvent systems. The intensity data for Me Sn(S2MoVa), Me Sn(S4MoVa) and
2
2
Me Sn(SBoVa) were measured at T = 150 K on an Oxford Diffraction Gemini E CCD diffractometer
2
(
(
Oxford Diffraction Ltd., Agilent Technologies, Santa Clara, CA, USA) fitted with Mo K
λ = 0.71073 Å) so that θmax was 29.4 . Data reduction, including analytical absorption correction, was
α
radiation
◦
accomplished with CrysAlisPro (Oxford Diffraction Ltd., UK) [78]. The structures were solved by direct
methods [79] and refined (anisotropic displacement parameters, C-bound H atoms in the riding model
2
2
2
2
2
2
approximation) on F . A weighting scheme w = 1/[
o
σ (F ) + (aP) + bP] where P = (Fo + 2Fc )/3) [80]
was introduced in each case. The crystal of Sn(S2MoVa)Me was refined as a twin with the fraction
2
of the minor component being 0.1664(20); details are given in the deposited CIF. In the final cycles of
the refinement of Me Sn(SBoVa), four reflections, i.e., (
−
3 4 0), (
omitted owing to poor agreement. The molecular structure diagrams was generated with ORTEP for
Windows [81] with 35, 50 and 70% displacement ellipsoids for Me Sn(S2MoVa), Me Sn(S4MoVa) and
−
1
−
1 5), (0 6 0) and (
−
3 4 4), were
2
2
2
Me Sn(SBoVa), respectively, and the packing diagrams were drawn with DIAMOND (Crystal Impact,
2
GbR, Bonn, Germany) [82]. Additional data analysis was carried out with PLATON [83]. Crystal data
and refinement details are given in Table 5.
3
.5. Computational Methods
3
.5.1. Density Functional Theory (DFT) Calculations
All DFT calculations were performed using Gaussian09 software (Gaussian Inc., Wallingford,
CT, USA) [84] and Gaussview5 program (Semichem, Inc., Shawnee Mission, KS, USA) [85] was
used to visualise the graphics. The molecular structures and geometries of the Schiff bases and
organotin(IV) compounds were fully optimised from the X-ray crystallographic structures using
density functional theory (DFT) method with the B3LYP [86,87] hybrid exchange correlation functional
with LanL2DZ pseudopotential on Sn [88 90] and 6-311G(d,p) Pople basis set for all other atoms.
–
Vibrational frequencies were calculated via the harmonic approximation to ensure that all optimised
structured corresponded to local minima of the potential energy surface. The electronic stabilities
of the optimised geometries were computed using the time-dependent density functional theory
(TD-DFT) formalism [91
,92] and included solvation effects (DMSO) via the polarisable continuum
method (PCM) [93–95], using the same basis set.

Int. J. Mol. Sci. 2019, 20, 854
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Table 5. Crystal data and refinement details for compounds Me Sn(S2MoVa), Me Sn(S4MoVa) and
2
2
Me Sn(SBoVa).
2
Compound
Me Sn(S2MoVa)
Me Sn(S4MoVa)
Me Sn(SBoVa)
2
2
2
Formula
Formula weight
Crystal colour
Crystal size/mm3
Crystal system
Space group
a/Å
C H N O S Sn
C H N O S Sn
C H N O S Sn
18 20 2 2 2
19
22
2
2
2
19 22
2
2
2
493.19
light-yellow
0.03 × 0.05 × 0.10
Monoclinic
Pn
493.19
479.19
light-yellow
0.05 × 0.13 × 0.20
Triclinic
P1
light-yellow
0.03 × 0.05 × 0.10
Triclinic
P1
11.3716(9)
12.0262(8)
16.3644(15)
90
109.285(9)
90
2112.4(3)
4
1.551
10.5908(4)
13.9243(5)
14.3974(5)
95.596(3)
93.693(3)
102.116(2)
2058.11(13)
4
8.9408(4)
10.4182(4)
22.6034(12)
97.713(4)
94.600(4)
111.634(4)
1920.11(16)
4
b/Å
c/Å
◦
α/
◦
β/
γ/
◦
V/Å3
Z
−
3
Dc/g cm
F(000)
1.592
992
1.46
1.658
960
1.562
992
1.422
−
1
µ(MoKα)/mm
Measured data
θ range/
Unique data
6348
3.6–29.4
6348
18090
3.5–29.4
9402
15229
3.4–29.4
8720
◦
Observed data (I ≥ 2.0σ(I))
5090
478
7605
477
6671
457
No. parameters
R, obs. data; all data
a; b in weighting scheme
Rw, obs. data; all data
GoF
0.037; 0.064
0.022; 0
0.055; 0.074
0.99
0.034; 0.070
0.026; 0.240
0.049; 0.078
1.01
0.041; 0.083
0.032; 0.781
0.062; 0.092
1.01
Range of residual electron
−
3
density peaks/eÅ
−0.56–0.66
−0.46–0.75
−1.03–0.63
3
.5.2. Molecular Docking Studies
The coordinates of B-DNA (PDB ID: 1BNA) dodecamer d(CGCGAATTCGCG) was obtained from
2
the Protein Data Bank (http://www.rcsb.org/pdb). The coordinates of the dimethyltin(IV) compounds,
Me Sn(S2MoVa), Me Sn(S4MoVa) and Me Sn(SBoVa) were taken from their crystal structures as a
2
2
2
Crystallographic Information File (CIF) and converted to the Protein Data Bank (PDB) format using
Mercury software (The Cambridge Crystallographic Data Centre, Cambridge, UK) [96], whereas the
coordinates for Ph Sn(S2MoVa), Ph Sn(S4MoVa) and Ph Sn(SBoVa) were obtained after minimisation
2
2
2
of energy using DFT method. Molecular docking studies were carried out using the AutoDock Tools
version 1.5.6 and the AutoDock version 4.2.5.1 program (Molecular Graphics Laboratory, La Jolla, CA,
USA) [97]. Water molecules were removed, polar hydrogen atoms and Kollman charges were added
to the receptor (DNA sequences). In the docking analysis, the binding site was assigned across all
of the minor and major grooves of the DNA molecule, which was enclosed in a 64
×
64
×
122 grid
box with a grid spacing of 0.375 Å. AutoDock was ran using the following parameters: 30 docking
trials, population size of 300, maximum number of energy evaluation ranges of 2,500,000, maximum
number of generations of 27,000, mutation rate of 0.02, cross-over rate of 0.8 and the default values
were used for other parameters. The parameter file of Autodock was modified to incorporate van der
Waals interactions and other required parameters for Sn obtained from the Autodock website [98].
The lowest binding energy of the conformation from the highest cluster was selected as the best binding
conformation between the ligand and receptor.

Int. J. Mol. Sci. 2019, 20, 854
26 of 34
3
.6. Biological Assays
3
.6.1. MTT Assays
The inhibitory effect of the synthesised compounds on the growth of bladder (EJ-28 and RT-112)
cancer cells was evaluated by MTT assay. The compounds were dissolved in DMSO and diluted
in culture medium, where the final concentration of DMSO did not exceed 0.05% (v/v) and was
not toxic to the cancer cells. RT-112 and EJ-28, human bladder cancer cell lines used in this study
(
1
ATCC, Manassas, VA, USA) were cultured in RPMI-1640 (High glucose) medium supplemented with
0% fetal bovine serum containing 1% penicillin. The cells were cultured at 37 C in a humidified
◦
atmosphere of 5% CO in air. Cells were seeded in 96-well plates at 6000 cells per well for 24 h, then
2
they were treated with different concentrations of compounds for 72 h. The medium was subsequently
removed and the wells were washed with 200
µL of phosphate buffer saline. Aliquots of 20 µL of
3
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well and
◦
incubated for 4 h at 37 C. Aliquots of 200 µL of media was then removed from each well and 200 µL
of DMSO was added. Optical density (OD) was measured using an ELISA plate reader at 570 nm.
Control wells (100% viability), containing media and DMSO, were included in all the experiments.
All data points represented an average of triplicate assays. Cytotoxicity was expressed as CD , i.e.,
50
the concentration that reduced the absorbance of treated cells by 50% with reference to the control
(untreated cells) [99].
For HT29 (colon), U87 and SJ-G2 (glioblastoma), MCF-7 (breast), A2780 (ovarian), H460 (lung),
A431 (skin), Du145 (prostate), BE2-C (neuroblastoma), MIA (pancreas) cell lines and one normal breast
cell line, MCF-10A (normal breast), the MTT assays were performed using the following method:
Cell Culture and Stock Solutions
◦
Stock solutions were prepared as follows and stored at
−
20 C: Trial compounds were stored
as 10 mM solutions in DMSO. All cell lines were cultured in a humidified atmosphere 5% CO
at 37 C. The cancer cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM)
2
◦
(
(
Trace Biosciences) supplemented with 10% foetal bovine serum, 10 mM sodium bicarbonate, penicillin
100 IU/mL), streptomycin (100 g/mL) and glutamine (4 mM). The normal cell line, MCF-10A was
µ
cultured in DMEM:F12 (1:1) cell culture media, 5% heat inactivated horse serum, supplemented with
penicillin (50 IU/mL), streptomycin (50 g/mL), 20mM Hepes, L-glutamine (2 mM), epidermal growth
µ
factor (20 ng/mL), hydrocortisone (500 ng/mL), cholera toxin (100 ng/mL) and insulin (10 µg/mL).
In Vitro Growth Inhibition Assay
Cells in logarithmic growth were transferred to 96-well plates. Cytotoxicity was determined by
plating cells in duplicate in 100
µL medium at a density of 2500–4000 cells/well. On day 0 (24 h after
plating), when the cells were in logarithmic growth, 100
µL medium, with or without the test agent,
was added to each well. After 72 h drug exposure, growth inhibitory effects were evaluated using the
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and absorbance read at
5
40 nm. Percentage growth inhibition was determined at a fixed drug concentration of 25 µM. A value
of 100% was indicative of complete cell growth inhibition. Those analogues showing appreciable
percentage growth inhibition underwent further dose-response analysis allowing for the calculation of
a GI50 value. This value is the drug concentration at which cell growth is 50% inhibited based on the
difference between the optical density values on day 0 and those at the end of drug exposure [100,101].
3
.6.2. Quantification of Apoptosis by Annexin V
The apoptotic death of RT-112 cells was measured by fluorescence microscopy using Annexin
V/PI double-staining. To observe the effect of the diphenyltin(IV) compounds on cell apoptosis, RT-112
6
cells, which are minimum invasive bladder cancer cells were seeded in 6-well plates at 1 × 10 cells
per well. After growth overnight, the inhibition concentration obtained from the CD50 analysis was

Int. J. Mol. Sci. 2019, 20, 854
27 of 34
used to treat the cells. After 24 h, the cells were washed with phosphate buffer saline (PBS) and 100 µL
of Annexin-V-FLUOS labelling solution containing the incubation buffer and then, propidium iodide
◦
(
PI) solution was added. The plate was then incubated for 10–15 min at 15–25 C and analysed by
fluorescence microscopy with a Fluorescence Inverted Microscope (Olympus IX51, Waltham, MA,
USA). Green staining of the plasma membrane indicated that the cells had bound to Annexin-V.
The cells that showed red staining were considered to have lost their membrane integrity. The cells
stained green were concluded to be apoptotic cells; the cells with both green and red staining were
concluded as late apoptotic cells whereas the cells that showed only red staining were considered as
necrotic cells [102].
3
.6.3. Measurement of Reactive Oxygen Species (ROS)
Intracellular ROS measurements were used to assess antioxidant or ROS activity. Measurements
were performed using a fluorescence plate reader following to the procedure outlines in the ROS
detection kit (OxiSelect
™
). In brief, RT-112 cells were cultured in 100
µ
L growth medium in a 96-well
◦
cell culture plate and incubated in an atmosphere of 5% CO at 37 C for a period of 24 h. The growth
2
medium was then removed, the cells were loaded with an oxidation sensitive cell-permeable fluorescent
probe, dichloro-dihydro-fluorescein diacetate (DCFH-DA), and subsequently stabilised in the highly
reactive DCFH form. In this reactive state, ROS species in living cells were reacted with DCFH, which
0
0
is rapidly oxidized to the highly fluorescent 2 ,7 -dichlorodihydrofluorescein (DCF). The plates were
then incubated for 1 h and the growth medium was removed. The cells in each well were washed
with PBS and the growth medium containing 3.05
DMSO), or positive control (H O ) was added. The plates were incubated in an atmosphere of 5% CO
2
µM of Ph Sn(S4MoVa) compound, negative control
2
(
2
2
◦
at 37 C for a period of 24 h. After incubation, the cells were transferred to black 96-well plates with
the addition of 2 Cell Lysis Buffer. The cells were then read on a standard Infinite M200 fluorescence
microplate reader (Tecan, Männedorf, Switzerland). The ROS or antioxidant content in unknown
samples was determined by comparison with the predetermined DCF standard curve [103].
×
3
.6.4. DNA Binding Studies
DNA binding experiments were carried out at 25 C. DNA concentration per nucleotide was
◦
−
1
−1
determined using the molar absorption coefficient (6600 M cm ) at 260 nm [
8
,104]. Solutions of
calf thymus (CT) DNA in Tris-HCl buffer containing (5 mM Tris, pH 7.2, 50 mM NaCl) [
8] showed
A260/A280 of 1.9, indicating that the DNA was sufficiently free of proteins and contaminants [105].
Absorption titration experiments were performed maintaining the concentration of the organotin(IV)
compounds solutions at 50
µM and gradually increasing the concentration of CT-DNA. The
organotin(IV) compounds were dissolved in DMSO and diluted with Tris-HCI buffer at room
◦
temperature (25 C). The solutions were scanned over 230–600 nm. Absorbance values were recorded 10
min after the addition of DNA solution. The binding constant, K was determined using the equation:
b
[
DNA]
εa − εf)
[DNA]
1
=
+
(
(ε − ε ) K (ε − ε )
b
f
b
b
f
where [DNA] is the concentration of DNA in base pairs, εa corresponds to the apparent molar extinction
coefficient Aabs/[M], corresponds to the extinction coefficient for the free organotin [M] and ε
ε
f
b
corresponds to the extinction coefficient for the fully bound organotin compound.
4
. Conclusions
Organotin(IV) compounds derived from S-substituted dithiocarbazates and o-vanillin were
synthesised and characterised by elemental analysis and various spectroscopic techniques (UV-visible,
1
13
119
FTIR, H, C and Sn NMR). The molecular structures of Me Sn(S2MOVa), Me Sn(S4MOVa) and
2
2
Me Sn(SBOVa) were elucidated by single crystal X-ray structure analysis which indicated that the
2

Int. J. Mol. Sci. 2019, 20, 854
28 of 34
compounds had five-coordinate C NOS geometries, derived from thiolate-S1, phenoxide-O1 and
2
imine-N atoms of a dinegative, tridentate dithiocarbazate ligand along with two methyl-carbon
atoms of the methyl groups, which were intermediate between ideal trigonal-bipyramidal and
square-pyramidal. The in vitro cytotoxicity against a panel of cancer cell lines viz., EJ-28 and RT-112
(
bladder), HT29 (colon), U87 and SJ-G2 (glioblastoma), MCF-7 (breast), A2780 (ovarian), H460 (lung),
A431 (skin), Du145 (prostate), BE2-C, (neuroblastoma) and MIA (pancreas) cancer cell lines revealed
the remarkable cytotoxicity of diphenyltin(IV) compounds for most of the cell lines, which were more
potent than cisplatin. In order to evaluate the mechanism of death of the organotin(IV) compounds,
Annexin-V and ROS assays were performed using the RT-112 cell line where the diphenyltin(IV)
compounds were able to induce apoptosis and ROS generation in the cells. The DNA binding of
the organotin(IV) compounds were investigated using UV-vis absorption and the diphenyltin(IV)
5
6
compounds bound reasonably well into the groove of DNA with binding constants in the range 10 –10
−
1
M
. The mode of interaction of the organotin(IV) compounds with DNA was further validated by
molecular docking studies which were in accordance with the experimental results, suggesting the
strong binding of diphenyltin(IV) at the AT-rich region of the minor groove of DNA. The observed
trend in the cytotoxicity and binding interactions of organotin(IV) compounds were attributed to the
nature of the group bound to the Sn(IV) metal centre where the diphenyltin(IV) compounds exhibited
potent efficacy and were promising leads for cytotoxic drug development.
Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/20/4/854/s1.
Crystallographic data for Me Sn(S2MoVa), Me Sn(S4MoVa) and Me Sn(SBoVa) reported in this paper have
2
2
2
been deposited with the Cambridge Crystallographic Data Centre (CCDC) as supplementary publication nos
1
834057-1834059. These data can be obtained free of charge via www.ccdc.cam.ac.uk/getstructures.
Author Contributions: Conceptualization, T.B.S.A.R.; Formal analysis, E.N.M.Y., M.I.S., A.J.P. and E.R.T.T.;
Funding acquisition, A.V. and T.B.S.A.R.; Investigation, E.N.M.Y., J.A.S., M.I.S., A.J.P. and A.V.; Methodology, A.V.
and E.R.T.T.; Project administration, J.A.S. and T.B.S.A.R.; Resources, A.V. and T.B.S.A.R.; Software, M.A.M.L.,
A.J.P. and E.R.T.T.; Supervision, M.I.M.T., M.I.S., A.J.P., A.V. and T.B.S.A.R.; Validation, M.A.M.L., A.J.P. and
E.R.T.T.; Visualization, A.J.P. and E.R.T.T.; Writing—original draft, E.R.T.T.; Writing—review & editing, M.I.S.,
A.J.P., A.V. and T.B.S.A.R.
Funding: This research was funded by Universiti Putra Malaysia under the Geran Putra IPS (9504600) and Geran
Putra IPB (9581001) (UPM) and the Malaysian Fundamental Research Grant Scheme (FRGS No. 01-01-16-1833FR).
Acknowledgments: We thank the Department of Chemistry, the Molecular Genetics Laboratory and the
Department of Biomedical Sciences, Universiti Putra Malaysia, Malaysia and the Discipline of Chemistry,
University of Newcastle and the Calvary Mater Hospital, Australia for their facilities. E.N.M.Y. wishes to
thank Ministry of Higher Education Malaysia for the award of MyPhD, MyBrain15 and University of Newcastle
for the award of University of Newcastle International Postgraduate Research Scholarship (UNIPRS), University
of Newcastle Research Scholarship Central (UNRSC) and Karen A. Crouse for helpful discussions.
Conflicts of Interest: The authors declare no conflict of interest.
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